FIG. 5.

Effect of GST-Ku80C in a cell-free transcription system. AdMLP template was preincubated with HeLa nuclear extract in the presence and absence of approximately 100 ng of GST-Ku80C or GST control protein for 30 min at 30°C. Transcription was initiated by addition of a mixture of ATP, UTP, and [α-³²P]CTP. An arrowhead denotes the 380-nucleotide full-length AdMLP transcript. RNA was analyzed by urea-PAGE and visualized by PhosphorImager analysis. (A) Transcription assay performed under single and multiple round conditions. Where indicated, 10 μg of heparin per ml was added after initiation to prevent the initiation of additional rounds. (B) Reversal of inhibition by full-length Ku. Reactions were performed as in panel A, except that Ku heterodimer (prepared as in reference 71) and GST-Ku80C were incubated with nuclear extract for 10 min at 0°C prior to addition of template. (C) Lack of effect of full-length Ku alone. Reactions were performed in panel A in the presence and absence of approximately 100 ng of each of the indicated proteins.